I propose to investigate determinants of substrate specificity of several protein kinases that mediate the actions of the intracellular second messengers cAMP, cGMP, and calcium ion. Synthetic peptides and purified proteins will be used as model substrates and inhibitors in in vitro enzymological studies of purified cAMP-dependent cGMP-dependent, and multifunctional Ca2+/calmodulin-dependent protein kinases. Synthetic oligopeptides corresponding to amino acid sequences in the active portion of the heat-stable inhibitor protein of cAMP-dependent protein kinase will be used to investigate the specificity of this inhibitor for the two cyclic nucleotide-dependent protein kinases. An active catalytic fragment of cGMP-dependent protein kinase will be generated from the holoenzyme by limited proteolysis. The activity of this fragment will be compared to that of intact cGMP-dependent protein kinase to investigate the influence of the regulatory domain of the holoenzyme on the interaction of peptide substrates and inhibitors at the active site. In other studies, I will characterize the phosphorylation of cardiac muscle C-protein, a component of the thick-filament, by cGMP-dependent, cAMP-dependent, and Ca2+/calmodulin-dependent protein kinases with respect to kinetic parameters, stoichiometries and primary structure at sites of phosphorylation. Results will allow development of a method for mapping site-specific phosphorylation of C-protein from intact tissues under physiological conditions in which different protein kinases are activated. Knowledge of phosphorylation sites in C-protein will also aid structure-function studies of purified C-protein selectively phosphorylated by different protein kinases. The potential presence of C-protein or C-protein-like immunoreactivity in tissues rich in smooth muscle will be investigated. Immunoreactive smooth muscle proteins will be studied as potential substrates of protein kinases that may mediate relaxation of smooth muscle in response to physiological stimuli, particularly the cGMP-dependent protein kinase. These studies should provide new information about the molecular details that dictate the substrate specificity determinants of cAMP-dependent, cGMP-dependent, and Ca2+/calmodulin-dependent protein kinases. An increased understanding of the differences in the active sites of these protein kinases may allow the development of selective inhibitors of each enzyme. Such reagents would be useful pharmacological tools in the study of the physiological roles of protein kinases and their substrates.